作者は感謝して乳房がん研究振興財団と高度な医療研究財団のサポートを認めます。

ここで説明したすべてのメソッドは、ボストン子供病院制度バイオ セーフティ委員会によって承認されています。
1. セル準備
<ol><li>
KHOS A を融解し、-n 個のセル
	<ol>
<li>ウォーム アップ培地、すなわち、ダルベッコ修飾イーグル培 10% (巻/巻) 牛胎児血清 (FBS) と 1% (巻/巻) ペニシリン/ストレプトマイシン。</li>
		<li>液体窒素タンクからセルのセラム チューブを取る、暖かい水 37 ° C の水浴中にバイアルの底を浸し、解凍処理を加速するセラム チューブを軽く振る。汚染を避けるために水へのお問い合わせからセラム チューブのふたを保ちます。細胞を解凍すると、すぐに、70% エタノール溶液を噴霧し、バイアルを拭く、セラムを滅菌します。</li>
		<li>層流フードの極低温バイアルから細胞懸濁液をすぐに吸引し、優しく温めて 10 mL 培養液 (1.1.1 で説明) で中断します。5-7 分の約 200 x g で細胞を懸濁液を遠心します。</li>
		<li>培地 10 mL を温めて、細胞ペレットを再懸濁します、T75 フラスコに移します。軽く均等に 10 mL のピペットを使用してセルを中断する数回をピペットします。</li>
		<li>5% (巻/巻) CO2と加湿雰囲気 37 ° C の定温器にフラスコを配置します。細胞の少なくとも 12 時間の接続を許可します。</li>
		<li>80-100% 合流まで約 3-7 日間細胞を孵化させなさい。2-3 日毎の培養培地を変更します。</li>
	</ol>
</li>
	<li>
KHOS A を分割し、-n 個のセル
	<ol>
<li>フラスコから文化メディアを削除します。</li>
		<li>洗浄 5 mL 滅菌 PBS のセル (1 x) カルシウムとマグネシウム、非付着性のセルまたは分数を削除するなし。</li>
		<li>フラスコ、1 mL 0.05% トリプシン EDTA 溶液を追加し、簡単にトリプシン-EDTA が均等に分散されるようにフラスコを旋回します。</li>
		<li>37 ° C の定温器で 2 〜 3 分または室温で 3-5 分のフラスコを孵化させなさい。セルは切り上げし、切り離されたことを確認する約 400 倍の倍率での顕微鏡の下のセルを確認します。</li>
		<li>セルを取り外したら、一度培地 10 mL を追加し、そっとピペット 10 mL のピペットを使用して単一細胞懸濁液に細胞集塊を破るために数回上下中。</li>
		<li>細胞カウンターを使用してセルをカウントします。2 × 10 の6セルの密度または 1:4 の比率で新しい T75 フラスコに細胞懸濁液をシードします。</li>
		<li>QPI 実験のため播種する前に 80-100% の合流点に成長する細胞を許可します。</li>
	</ol>
</li>
	<li>
KHOS A シードと QPI の - n 個のセル
	<ol>
<li>シード KHOS A とセル カウンター カウント後培地 5 mL を 50,000 300,000 細胞/ウェルの密度で 6 ウェル プレートで - n 個のセル。 注: 播種密度は細胞増殖率およびイメージングの期間に依存するために < 100% 取得、イメージング中に合流。</li>
		<li>添付ファイルを許可するように少なくとも 12 時間のセルを孵化させなさい。</li>
		<li>QPI を行う前に新鮮なメディア媒体を変更します。</li>
	</ol>
</li>
</ol>2. QPI 買収
<ol><li>
カバー スリップを設定
	<ol>
<li>脱イオン水を実行して数回の洗浄によってカバー スリップをきれいにし、カバー スリップを入れ乾燥滅菌層流フード 15 分の浸漬で 75% エタノール溶液で消毒します。</li>
		<li>明白な泡を避けるために 6 ウェル プレート カバー スリップを慎重に配置します。カバー スリップの下が文化メディア (最小 5 mL/ウェル) に没頭していることを確認します。</li>
		<li>(37 ° C、5% CO2、および加湿雰囲気) 少なくとも 15 分平衡にインキュベーターにプレートを配置します。カバー スリップ上に霧を形成、それをきれいに拭くため滅菌綿棒を使用します。</li>
	</ol>
</li>
	<li>
顕微鏡で細胞のイメージング
	<ol>
<li>露出時間、パターン コントラスト、およびホログラム ノイズの自己校正を含むシステムを初期化するソフトウェアを開きます。値が許容される (緑または黄色の範囲で表示) を確認します。</li>
		<li>QPI 顕微鏡のステージ上に 6 ウェル プレートを配置します。</li>
		<li>「Live Capture"をクリックしてし「ソフトウェア フォーカス」で「手動」を選択「顕微鏡の設定」の作業距離を調整することによって細胞の位相画像を粗く集中位相画像のセルのアウトラインを取得しますします。「ソフトウェア フォーカス」で、「自動」に変更します。</li>
		<li>「新しい実験」をクリックして新しいテストを作成します。テンキー、マウスまたは矢印キーを使用してイメージの取得位置の選択を開始します。各選択した後「保存」をクリックします。通常の各サンプルに対して 3-5 場所が選択されます。</li>
		<li>「タイムラプス」セクションのイメージの間隔と期間を設定します。 注: がん細胞を明確に追跡するには、間隔が 5 分 48 時間は組み合わせ QPI 分析の期間として設定より短いでする必要があります。</li>
		<li>「キャプチャ」をクリックして自動的に焦点を当て、設定の時点で画像を取得する実験を開始します。</li>
	</ol>
</li>
</ol>3. データ分析
<ol><li>
細胞の形態解析
	<ol>
<li>「細胞を識別」をクリックバック グラウンド ノイズからのセル領域が分離できるように、「背景のしきい値」の数値の設定を調整します。「オブジェクト サイズ」の設定数を調整それぞれセルに 1 つの核を確認しますします。比較する必要がある最終的な面積と体積の測定に影響を与える可能性がありますこれらのサンプルのための一貫したパラメーターを維持します。手動で必要な場合、「手動で変更、」を使用して、セル セグメントを変更します。</li>
		<li>ソフトウェアの「データの分析」関数を使用して、セル分析。「新分析」をクリックしてして新しいデータ解析をを開始します。セル領域 (2μ m)、光学膜厚 (μ m)、ボリューム (μ3) すべての個々 のセルに対してなど「ソース フレーム」タブと細胞形態のパラメーターに選択した画像をドラッグします。表や図としての散布図のプロットやヒストグラム モードとエクスポート データを選択します。</li>
	</ol>
</li>
	<li>
細胞増殖の解析
	<ol>
<li>(例えば初期、12 h、24 h、36 h、および 48 h) 興味の別の時間ポイントの少なくとも 5 枚の画像を選択します。ソフトウェアによって提供されるレコードの携帯番号。</li>
		<li>倍加時間を推定するには、初期の番号の正規化後細胞数をプロットし、指数関数的成長曲線にフィットします。</li>
	</ol>
</li>
	<li>
細胞運動解析
	<ol>
<li>ソフトウェアの「細胞を追跡する」関数を使用します。「新分析」をクリックして新しいデータ分析を開始します。(例えば、24 時間培養後 4 h)、「ソース フレーム」タブに選択した時間帯のイメージのドラッグ シリーズ ランダムに「セルを追加」選択モード下のセルをクリックしてして 10-30 セルを選択します。エッジとそれらのビューのフィールドの外に移動セルを除外します。</li>
		<li>一連の画像でトラッキングを入念にチェックします。システムはセルのトラックを失うまたは誤ったセルを追跡、こと手動でセルの位置を変更するのには「選択」モードの下で追跡携帯を調整細胞を識別するために「識別する」をクリックするか、「場所の変更」をクリック。</li>
		<li>「プロット運動」におけるセル軌道バラ プロットまたは移行率直さ、移行 (μ m) (μ m) の運動運動速度 (μ m/h) などの「印刷機能」で、そのほかの運動関連パラメーターのプロットを選択します。データを表や図としてエクスポートします。</li>
	</ol>
</li>
</ol>

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<li>Hu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+signature+for+angiogenic+and+nonangiogenic+non-small-cell+lung+cancer%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Gene expression signature for angiogenic and nonangiogenic non-small-cell lung cancer.</a> Oncogene. 24 (7), 1212-1219 (2005).</li>
<li>Harper, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repression+of+vascular+endothelial+growth+factor+expression+by+the+zinc+finger+transcription+factor+ZNF24%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Repression of vascular endothelial growth factor expression by the zinc finger transcription factor ZNF24.</a> Cancer Research. 67 (18), 8736-8741 (2007).</li>
<li>Jia, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+repression+of+VEGF+by+ZNF24%3A+mechanistic+studies+and+vascular+consequences+in+vivo%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Transcriptional repression of VEGF by ZNF24: mechanistic studies and vascular consequences in vivo.</a> Blood. 121 (4), 707-715 (2013).</li>
<li>Jia, D., Huang, L., Bischoff, J., Moses, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+endogenous+zinc+finger+transcription+factor%2C+ZNF24%2C+modulates+the+angiogenic+potential+of+human+microvascular+endothelial+cells%5BTitle%5D)+AND+%22FASEB+journal%3A+official+publication+of+the+Federation+of+American+Societies+for+Experimental+Biology%22%5BJournal%5D)">The endogenous zinc finger transcription factor, ZNF24, modulates the angiogenic potential of human microvascular endothelial cells.</a> FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 29 (4), 1371-1382 (2015).</li>
<li>Almog, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prolonged+dormancy+of+human+liposarcoma+is+associated+with+impaired+tumor+angiogenesis%5BTitle%5D)+AND+%22FASEB+journal%3A+official+publication+of+the+Federation+of+American+Societies+for+Experimental+Biology%22%5BJournal%5D)">Prolonged dormancy of human liposarcoma is associated with impaired tumor angiogenesis.</a> FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 20 (7), 947-949 (2006).</li>
<li>Almog, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Consensus+micro+RNAs+governing+the+switch+of+dormant+tumors+to+the+fast-growing+angiogenic+phenotype%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Consensus micro RNAs governing the switch of dormant tumors to the fast-growing angiogenic phenotype.</a> PloS One. 7 (8), e44001(2012).</li>
<li>Satchi-Fainaro, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prospective+identification+of+glioblastoma+cells+generating+dormant+tumors%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Prospective identification of glioblastoma cells generating dormant tumors.</a> PloS One. 7 (9), e44395(2012).</li>
<li>Almog, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+changes+induced+by+the+tumor+dormancy-associated+microRNA-190%5BTitle%5D)+AND+%22Transcription%22%5BJournal%5D)">Transcriptional changes induced by the tumor dormancy-associated microRNA-190.</a> Transcription. 4 (4), 177-191 (2013).</li>
<li>Gao, D., Nolan, D. J., Mellick, A. S., Bambino, K., McDonnell, K., Mittal, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+progenitor+cells+control+the+angiogenic+switch+in+mouse+lung+metastasis%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Endothelial progenitor cells control the angiogenic switch in mouse lung metastasis.</a> Science. 319 (5860), New York, N.Y. 195-198 (2008).</li>
<li>Folkman, J., Watson, K., Ingber, D., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+angiogenesis+during+the+transition+from+hyperplasia+to+neoplasia%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Induction of angiogenesis during the transition from hyperplasia to neoplasia.</a> Nature. 339 (6219), 58-61 (1989).</li>
<li>Popescu, G. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPopescu%2C+G+%22Quantitative+phase+imaging+of+cells+and+tissues%22">Quantitative phase imaging of cells and tissues</a>. , McGraw-Hill: New York. (2011).</li>
<li>Lee, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+Phase+Imaging+Techniques+for+the+Study+of+Cell+Pathophysiology%3A+From+Principles+to+Applications%5BTitle%5D)+AND+%22Sensors%22%5BJournal%5D)">Quantitative Phase Imaging Techniques for the Study of Cell Pathophysiology: From Principles to Applications.</a> Sensors. 13 (4), 4170-4191 (2013).</li>
<li>Mir, M., Bhaduri, B., Wang, R., Zhu, R., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+Phase+Imaging%5BTitle%5D)+AND+%22Progress+in+Optics%22%5BJournal%5D)">Quantitative Phase Imaging.</a> Progress in Optics. 57, 133-217 (2012).</li>
<li>Marrison, J., Räty, L., Marriott, P., O'Toole, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ptychography--a+label+free%2C+high-contrast+imaging+technique+for+live+cells+using+quantitative+phase+information%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Ptychography--a label free, high-contrast imaging technique for live cells using quantitative phase information.</a> Scientific Reports. 3, 2369(2013).</li>
<li>Falck Miniotis, M., Mukwaya, A., Gjörloff Wingren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Digital+holographic+microscopy+for+non-invasive+monitoring+of+cell+cycle+arrest+in+L929+cells%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Digital holographic microscopy for non-invasive monitoring of cell cycle arrest in L929 cells.</a> PloS One. 9 (9), e106546(2014).</li>
<li>Popescu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+imaging+of+cell+mass+and+growth+dynamics%5BTitle%5D)+AND+%22AJP%3A+Cell+Physiology%22%5BJournal%5D)">Optical imaging of cell mass and growth dynamics.</a> AJP: Cell Physiology. 295 (2), C538-C544 (2008).</li>
<li>Guo, P., Huang, J., Moses, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+dormant+and+active+human+cancer+cells+by+quantitative+phase+imaging%5BTitle%5D)+AND+%22Cytometry.+Part+A%3A+The+Journal+of+the+International+Society+for+Advancement+of+Cytometry%22%5BJournal%5D)">Characterization of dormant and active human cancer cells by quantitative phase imaging.</a> Cytometry. Part A: The Journal of the International Society for Advancement of Cytometry. 91 (5), 424-432 (2017).</li>
<li>Mir, M., Bergamaschi, A., Katzenellenbogen, B. S., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+sensitive+quantitative+imaging+for+monitoring+single+cancer+cell+growth+kinetics+and+drug+response%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Highly sensitive quantitative imaging for monitoring single cancer cell growth kinetics and drug response.</a> PloS One. 9 (2), e89000(2014).</li>
<li>Mir, M., Tangella, K., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood+testing+at+the+single+cell+level+using+quantitative+phase+and+amplitude+microscopy%5BTitle%5D)+AND+%22Biomedical+Optics+Express%22%5BJournal%5D)">Blood testing at the single cell level using quantitative phase and amplitude microscopy.</a> Biomedical Optics Express. 2 (12), 3259-3266 (2011).</li>
<li>Park, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+red+blood+cell+mechanics+during+morphological+changes%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Measurement of red blood cell mechanics during morphological changes.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (15), 6731-6736 (2010).</li>
<li>Pham, H. V., Bhaduri, B., Tangella, K., Best-Popescu, C., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real+time+blood+testing+using+quantitative+phase+imaging%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Real time blood testing using quantitative phase imaging.</a> PloS One. 8 (2), e55676(2013).</li>
<li>Sridharan, S., Macias, V., Tangella, K., Kajdacsy-Balla, A., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prediction+of+prostate+cancer+recurrence+using+quantitative+phase+imaging%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Prediction of prostate cancer recurrence using quantitative phase imaging.</a> Scientific Reports. 5, 9976(2015).</li>
<li>Park, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measuring+cell+surface+area+and+deformability+of+individual+human+red+blood+cells+over+blood+storage+using+quantitative+phase+imaging%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Measuring cell surface area and deformability of individual human red blood cells over blood storage using quantitative phase imaging.</a> Scientific Reports. 6, 34257(2016).</li>
<li>Bishitz, Y., Gabai, H., Girshovitz, P., Shaked, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical-mechanical+signatures+of+cancer+cells+based+on+fluctuation+profiles+measured+by+interferometry%5BTitle%5D)+AND+%22Journal+of+Biophotonics%22%5BJournal%5D)">Optical-mechanical signatures of cancer cells based on fluctuation profiles measured by interferometry.</a> Journal of Biophotonics. 7 (8), 624-630 (2014).</li>
</ol>

利害の対立が宣言されていません。

本研究生体外で、非侵襲的、無料のラベルを用いた QPI ひと骨肉腫細胞の血管新生と血管新生以外の表現型を特徴づける定量的をについて説明します。増殖率、倍加時間、移行の率直さ、運動速度、移行と運動細胞容積、セル厚セル領域を含むこの統合、ハイスループット法による細胞の複数のパラメーターを同時に分析されています。
細胞の形態や細胞の挙動を...

開発・進行固形腫瘍の初期のチェックポイントの 1 つは、血管新生形質、がんの特徴の習得です。この進行には、様々 な生化学的・分子過程1,2,3が含まれます。腫瘍の進行のこの重要なステップにおける技術的な課題は、継続的かつ定量的評価し、公平な方法で生きているがん細胞の血管新生と血管新生以外の表現型を区別するためのツールの欠如です。通常血管新生と非血管新生細胞の細胞の挙動を調査するために使用されている伝統的な試金は高価な試薬・機器を必要とする、たとえば、細胞増殖・遊走アッセイ4,5、 6,7,8,9,10、11,12,13,14か高い専門性と集中を必要とすると同様に相補的な生体内評価4,5,6,8,15,16、時間と労働消費。
最近では、定量位相イメージング (QPI) は、様々 な細胞形態および動作パラメーター17,18,19,の時間経過およびラベリング無料評価を可能にする手法として浮上しています。20,21,22. 従来の光学顕微鏡とは異なり QPI を定量化位相差画素ごとのバリエーション光、光物体を通り抜けるし、変換の光学的厚さとボリューム、自筆を再構築後、直通ができ生きているセル、次の機能の解析: (4) 同時多重パラメーター イメージング (3) ラベル無料画像、(2) 非侵襲とタイムラプス イメージング (1) 定量的イメージング。これらの機能は、QPI に評価し、細胞レベルで病態を理解するための強力なツールを作る。
最近の研究で我々 は定量的評価し、血管新生を区別する QPI を利用 KHOS A および体系的かつ定量的に、細胞形態の分析を組み合わせることでひと骨肉腫細胞の非血管新生 KHOS N の表現型増殖、および運動23。血管新生と血管新生以外のひと骨肉腫細胞の細胞形態および行動のパラメーターを比較した定量的画像解析ソフト、パネルを使用して、5 つの特徴的な違いは、これらの 2 つの識別されました。表現型。この手法は、さまざまな生物学的に関連する細胞の特性を評価するため統合と定量的なプラットフォームを提供します。

<table><tbody><tr><td>T75 flask</td><td>Corning, NY, USA</td><td>353136</td><td></td></tr><tr><td>6-well plates&amp;nbsp;</td><td>Corning, NY, USA</td><td>3506</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium (DMEM)</td><td>Thermo Fisher Scientific, MA, USA</td><td>11965092</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Atlanta Biologicals, GA, USA</td><td>S11550</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Thermo Fisher Scientific, MA, USA</td><td>15140122</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Thermo Fisher Scientific, MA, USA</td><td>10010023</td><td></td></tr><tr><td>Beckman Z1 Coulter counter</td><td>Beckman Coulter, IN, USA</td><td>Z1&amp;nbsp;</td><td></td></tr><tr><td>HoloMonitor M4</td><td>Phase Holographic Imaging Phi AB, Lund, Sweden</td><td>M4</td><td>Microscope</td></tr><tr><td>Hololid</td><td>Phase Holographic Imaging Phi AB, Lund, Sweden</td><td>PHI 8020</td><td></td></tr><tr><td>HStudioM4</td><td>Phase Holographic Imaging Phi AB, Lund, Sweden</td><td>HStudioM4</td><td>Software</td></tr></tbody></table>

a time-lapse, label-free, quantitative phase imaging study of dormant and active human cancer cells

血管新生形質の獲得は腫瘍休眠からの脱出の重要なコンポーネントです。これらのメソッドは時間を調査し、非血管新生と血管新生細胞の表現型を特徴付けるいくつかのクラシックの in vitroアッセイ (例えば増殖、移行、および他の人) と体内のモデルが開発されているが労働集約的なと、多くの場合高価な試薬、機器と専門的な技術を必要とします。最近の研究で血管新生と血管新生以外のひと骨肉腫 KHOS 細胞の時間経過およびラベリング-無料の特徴づけを行う新規定量位相イメージング (QPI) を使用しました。細胞の形態・増殖・運動を含む携帯電話のパラメーターのパネルは、定量的に測定したし、QPI を用いています。この小説と定量的アプローチは、継続的に、非侵襲的シンプルで統一された方法で関連する細胞プロセス、動作、およびがん細胞の特性および他の細胞型を勉強する機会を提供します。このレポートでは、セルの準備、QPI 集録、データ分析など当社の実験プロトコルについて説明します。

図 1は、典型的な細胞形態特性を示しています。画像は、ホログラフィー (図 1 aB) と 2次元画像 (図 1D) として掲載されています。(屈折率と光路長から計算) 光学セルの厚さは線またはセル全体の測定で定量化しました。散布図の面積と厚み KHOS A と図 1E<s...

Watch this Scientific Journal Video about A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells at JoVE.com

A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells

休止状態とアクティブな癌細胞の表現型は、定量的な位相画像を用いた特徴付けられました。細胞の増殖、移行、および形態の試金を統合し、1 つの単純な方法で分析します。

時間経過、ラベル無料、定量的相休眠とアクティブなひと癌細胞のイメージング研究

The acquisition of the angiogenic phenotype is an essential component of the escape from tumor dormancy. Although several classic in vitro assays (e.g., ...

a-time-lapse-label-free-quantitative-phase-imaging-study-dormant

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

7.4K ビュー.  Boston Children's Hospital. 休止状態とアクティブな癌細胞の表現型は、定量的な位相画像を用いた特徴付けられました。細胞の増殖、移行、および形態の試金を統合し、1 つの単純な方法で分析します。

ビデオ: A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

アンインビトロ</em転移性増殖に腫瘍の休眠とスイッチを研究する>システム

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

医学

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

長期的な住セルイメージ投射細胞の運命をパクリタ キセルに応答を評価するために

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

がん研究

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

侵襲性外科生検に対する脳腫瘍細胞行動の縦腔内イメージング

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

M1マクロファージ-4T1マウス乳腺癌細胞における食細胞活性のタイムラプス2Dイメージング

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

乳がんの遺伝子改変マウスモデル由来の初代前癌乳腺上皮細胞のタイムラプスイメージング

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

生物学

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

鏡の国のアリス：単一細胞のタイムラプス顕微鏡と縦トラッキング抗がん治療薬を研究します

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

腫瘍微小環境におけるリアルタイム多細胞ダイナミクスの拡張タイムラプス生体内イメージング

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

タイムラプスビデオ顕微鏡を用いた細胞のランダムな移行の定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification&#160;and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

水動紡錘摂動後のメタフェイズタイミングと細胞運命のダイナミクスを評価するライブ細胞イメージング

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...

時間経過、ラベル無料、定量的相休眠とアクティブなひと癌細胞のイメージング研究

要約

さらに動画を探す

アンインビトロシステム

長期的な住セルイメージ投射細胞の運命をパクリタキセルに応答を評価するために

侵襲性外科生検に対する脳腫瘍細胞行動の縦腔内イメージング

M1マクロファージ-4T1マウス乳腺癌細胞における食細胞活性のタイムラプス2Dイメージング

乳がんの遺伝子改変マウスモデル由来の初代前癌乳腺上皮細胞のタイムラプスイメージング

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

鏡の国のアリス：単一細胞のタイムラプス顕微鏡と縦トラッキング抗がん治療薬を研究します

腫瘍微小環境におけるリアルタイム多細胞ダイナミクスの拡張タイムラプス生体内イメージング

タイムラプスビデオ顕微鏡を用いた細胞のランダムな移行の定量分析

水動紡錘摂動後のメタフェイズタイミングと細胞運命のダイナミクスを評価するライブ細胞イメージング

時間経過、ラベル無料、定量的相休眠とアクティブなひと癌細胞のイメージング研究

要約

さらに動画を探す

アンインビトロシステム

長期的な住セルイメージ投射細胞の運命をパクリタ キセルに応答を評価するために

侵襲性外科生検に対する脳腫瘍細胞行動の縦腔内イメージング

M1マクロファージ-4T1マウス乳腺癌細胞における食細胞活性のタイムラプス2Dイメージング

乳がんの遺伝子改変マウスモデル由来の初代前癌乳腺上皮細胞のタイムラプスイメージング

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

鏡の国のアリス：単一細胞のタイムラプス顕微鏡と縦トラッキング抗がん治療薬を研究します

腫瘍微小環境におけるリアルタイム多細胞ダイナミクスの拡張タイムラプス生体内イメージング

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長期的な住セルイメージ投射細胞の運命をパクリタキセルに応答を評価するために